Tissue Factor–Factor VIIa Signaling

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     From the Biomedical Research Division, The University of Texas Health Center at Tyler, Tex.

    Correspondence to L. Vijaya Mohan Rao, PhD, Biomedical Research, The University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, TX 75708. E-mail vijay.rao@uthct.edu

    Abstract

    How does tissue factor (TF), whose principle role is to support clotting factor VIIa (FVIIa) in triggering the coagulation cascade, affect various pathophysiological processes? One of the answers is that TF interaction with FVIIa not only initiates clotting but also induces cell signaling via activation of G-protein–coupled protease activated receptors (PARs). Recent studies using various cell model systems and limited in vivo systems are beginning to define how TF–VIIa-induced signaling regulates cellular behavior. Signaling pathways initiated by both TF–VIIa protease activation of PARs and phosphorylation of the TF–cytoplasmic domain appear to regulate cellular functions. In the present article, we review the emerging data on the mechanism of TF-mediated cell signaling and how it regulates various cellular responses, with particular focus on TF–VIIa protease-dependent signaling.

    Recent studies show that tissue factor–factor VIIa, whose primary function is to initiate the clotting cascade, transduces cell signaling in various cell types. This brief review summarizes recent literature on potential mechanisms by which tissue factor–factor VIIa activates cell signaling, and how tissue factor–factor VIIa-induced cell signaling may affect various pathophysiological processes.

    Key Words: tissue factor ? factor VIIa ? protease activated receptors ? cell signaling

    Introduction

    The close link between coagulation and various diseases, such as sepsis, atherosclerosis, and tumor metastasis, suggests a complex interplay between the clotting cascade and disease progression. Numerous studies have demonstrated that coagulation proteases can function like hormones to regulate cellular behavior. For example, thrombin, the principal protease generated during coagulation, has been shown to activate platelets and regulate the behavior of other cells by transmitting signals via activation of G protein-coupled protease activated receptors (PARs). Although there has been initial skepticism on whether other clotting proteases can also activate PARs, recent studies provide convincing evidence that many proteases involved in clotting can indeed activate PARs and regulate cellular behaviors at physiological concentrations. One such protease, FVIIa, the physiological initiator of the coagulation cascade, has received much attention lately. The focus of the present article is to review briefly recent developments in tissue factor (TF)–VIIa proteolytic activity-mediated cell signaling.

    TF–Factor VIIa

    TF is a transmembrane cellular receptor for FVII/FVIIa. Binding of plasma FVII/FVIIa to TF triggers the coagulation cascade, which leads to thrombin generation that subsequently stimulates platelet activation and cleaves fibrinogen. Under normal conditions, TF is constitutively expressed in many cell types, including fibroblasts and pericytes in and surrounding blood vessel walls, but is not expressed in blood cells or the endothelial cells that line blood vessels.1,2 However, under certain pathological conditions, such as sepsis and cancer, monocytes3 and endothelial cells4 express TF, although the latter finding has not been confirmed by others.5 Thus, blood vessel wall injury or certain disease conditions permit FVII/FVIIa interaction with TF on cell surfaces. The activation of TF-induced coagulation pathway not only leads to fibrin formation but also contributes to vascular remodeling, which is caused by growth factors secreted by activated platelets, as well as the intermediary products factor Xa and thrombin that promote vascular smooth cell proliferation6,7 and alter endothelium.8 TF expression has been linked directly to various pathophysiological processes, such as development, inflammation, and tumor metastasis.9–11

    PARs

    Certain proteases control cellular functions via signaling through specific membrane receptors known as PARs, members of a larger family of 7 transmembrane cell surface receptors that mediate cell activation via G-proteins. At present, there are 4 known PARs: PAR1, PAR2, PAR3, and PAR4. PAR1, the prototypic family member, is cleaved primarily by thrombin, but can also be cleaved by other proteases, such as FXa,12 activated protein C (APC),13 plasmin,14 and FVIIa.15 PAR3 and PAR4 are also cleaved by thrombin, whereas PAR2 is cleaved by the trypsin-like enzymes, FXa16 and FVIIa.15 Although the mechanism of PARs activation was initially established for thrombin and PAR1, the mechanism is similar for the other PARs. In short, the protease cleaves the receptor at a specific site in the amino-terminal extracellular domain, leading to the unmasking of a new N-terminus. The new N-terminus then acts as a tethered ligand, binding intramolecularly to the body of the receptor to initiate transmembrane signaling. In fact, synthetic peptides mimicking the tethered ligand can activate the receptor independent of its cleavage. A number of excellent reviews provide details of the mechanism of activation of PARs and define the role for these receptors in vivo.17–19

    TF–VIIa-Induced Signaling

    Because aberrant expression of TF is associated with various diseases and TF is structurally similar to members of the class 2 cytokine receptor family,20 there has been a great interest in examining the role of TF in cell signaling. The first evidence for TF–VIIa-induced signaling came from studies in which FVIIa binding to TF was shown to induce intracellular Ca2+ oscillations in a number of TF-expressing cells.21,22 This FVIIa-induced calcium signaling required the binding of catalytically active FVIIa to TF but did not require the presence of TF cytoplasmic domain. Although cells that express TF, either inducibly or constitutively, responded to FVIIa binding, the fraction of cells that released Ca2+ and the extent to which Ca2+ was released varies among different cell types.23 Interestingly, some cell lines, such as HK-2 (a kidney cell line that constitutively expresses TF) and CHO cells stably transfected with TF, failed to respond to FVIIa, despite abundant expression of TF on their surfaces.24

    Work from other laboratories demonstrated that FVIIa interaction with TF on a variety of cells, including fibroblasts, epithelial cells, and endothelial cells, activated multiple signaling pathways. Poulsen et al25 reported first that FVIIa binding to BHK cells that are stably transfected with human TF [BHK(TF)] resulted in a transient activation of p44/42 MAPK. Further studies showed that the FVIIa-induced p44/42 MAPK activation is dependent on proteolytically active FVIIa, but independent of the TF cytoplasmic domain.26 FVIIa is shown to activate p44/42 MAPK in other cell types that express TF, such as fibroblasts27 and keratinocytes,28 but the response is not as robust as that observed in BHK(TF) cells. Additionally, FVIIa treatment of keratinocytes also increased the phosphorylation of key components of the other 2 MAPK pathways, p38 and C-Jun N-terminal kinase (JNK).24 Versteeg et al29 demonstrated that FVIIa stimulates a signaling pathway in fibroblasts (A14 cells), leading to the activation of the Src-like family members c-Src, Lyn, and Yes, and subsequently PI3-kinase, which then induces the stimulation of p44/42 MAPK, c-Akt/protein kinase B, and the small GTPases Rac and Cdc42. This group also showed that FVIIa-induced p44/42 MAPK activation is mediated via p21 Ras activation in BHK(TF) and HaCaT keratinocytes.30 In HaCaT cells, FVIIa has been shown to phosphorylate PYK2,31 a kinase that has been implicated in the regulation of MAP kinase activation.32 In addition to activating the p21 ras/MAPK pathway, FVIIa has also been shown to induce STAT5 phosphorylation via Jak2 activation in BHK(TF) cells33 and to stimulate the protein synthesis machinery via activation of p70/p85s6K, p90RSK, and eventually eukaryotic initiation factor eIF-4E.34 Although the described data provide convincing evidence that TF–VIIa activates multiple signaling pathways that could affect various cellular processes, one should exercise caution in extrapolating these data because the majority of these data were derived from a single cell line, BHK(TF).

    To address how TF-mediated cell signaling could potentially contribute to various pathophysiological conditions, several groups have focused on investigating TF–VIIa-induced alterations in gene expression. Examination of specific gene transcripts, whose products were believed to be pathophysiologically relevant to diseases associated with aberrant expression of TF, revealed that exposure of TF-expressing cells (fibroblasts, tumor cells, or keratinocytes) to FVIIa led to increased expression of vascular endothelial growth factor (VEGF),35 uPAR,36 Egr-1,24 and IL-8.37,38 Global analysis of TF–VIIa protease-induced signaling on the transcriptional machinery revealed that FVIIa binding to TF on fibroblasts or keratinocytes alters the expression of a few select genes. One of the upregulated transcripts observed in differential display polymerase chain reaction was identified as a poly(A) polymerase, whose product plays an important role in the processing of mRNA. Microarray analysis of fibroblasts briefly exposed to FVIIa revealed that FVIIa upregulates the expression of Cyr61 (CCN1) and connective tissue growth factor (CCN2).39 CCN1 and CCN2 are extracellular matrix signaling proteins that were recently shown to regulate a myriad of cellular functions, such as cell adhesion, proliferation, migration, and tumor metastasis.40 Using low-density cDNA arrays, Camerer et al28 showed that FVIIa interaction with TF on keratinocytes upregulates several genes that are relevant to the wound repair process.

    TF–VIIa Activation of a PAR

    Because TF–VIIa-induced signaling requires catalytically active FVIIa and is independent of the TF cytoplasmic tail, it has been hypothesized that TF–VIIa transmits cell signaling via PAR activation. However, until recently there was confusion about whether TF–VIIa mediated the cell signaling via 1 of the 4 known PARs or involved the activation of a novel PAR. The lack of heterologous desensitization (ie, failure of PAR agonists to abolish the response induced by subsequently added VIIa or vice versa), the differences between PAR agonists and FVIIa for their ability to induce Ca2+ release and p44/42 MAPK activation, and the lack of FVIIa-induced response in cells that express TF and the known PARs indicated that FVIIa may induce intracellular signaling via activation of a novel PAR or requires, in addition to a known PAR, an additional cell surface component.21,24,26,39,41 However, a recent study by Camerer et al15 showed that FVIIa induces Ca2+ release in Xenopus oocytes transfected with TF together with PAR1 or PAR2, but not PAR3 and PAR4. Similarly, transfection of lung fibroblasts from PAR1-deficient mice with TF and PAR2 conferred the FVIIa-induced response.15 These data suggest that TF–VIIa activates PAR2 and, to a lesser degree, PAR1. This conclusion is further supported by the observation that FVIIa can induce p44/42 MAPK activation in CHO cells transfected with TF and PAR2 but not in CHO cells expressing TF alone.42 Additionally, recent data38 demonstrate that specific antibodies against PAR2 but not PAR1 block FVIIa-induced IL-8 gene expression and cell migration in breast carcinoma cells. Similarly, PAR2 antibodies were shown to block TF–VIIa-induced smooth muscle cell migration.43 Together, these data suggest that TF–VIIa transmits cell signaling via activation of PAR2 (Figure 1). However, this does not mean that TF–VIIa cannot transmit signals via receptors other than PAR2.

    Figure 1. A schematic representation of TF–VIIa protease-induced signaling. TF–VIIa activates PAR2 and the ternary complex of TF–VIIa–Xa activates both PAR1 and PAR2. Activation of PAR2-specific signaling pathway may lead to phosphorylation of TF cytoplasmic tail. Phosphorylation of TF cytoplasmic domain releases its negative regulatory control of PAR2-mediated signaling.

    At present, it is unclear why FVIIa fails to induce calcium signaling in a number of cell types, including fibroblasts, epithelial cells, and tumor cells that express abundant TF and one or more PARs (PAR1 and PAR2)27,38,41 (and also unpublished data of the authors), and respond robustly to FVIIa stimulation by activating p44/42 MAPK25,41 or gene expression.39 In fact, failure to induce Ca2+ release and the lack of the desensitizing effect of FVIIa on PAR1 and PAR2 agonist-induced Ca2+ release lead to a conclusion, which in retrospective is not well-justified, that TF–VIIa-induced intracellular signaling is not mediated via known PARs but may involve proteolytic cleavage of an unidentified member of the PAR family.41 Why does activation of PAR2/PAR1 by TF–VIIa, in contrast to their activation by thrombin, trypsin, or PAR agonist peptides, fail to induce Ca2+ mobilization? One possible explanation may be an inefficient, but physiologically important, cleavage of PAR2/PAR1 by TF–VIIa. Earlier studies44 have shown that the magnitude of the PAR1-mediated protease response is determined by both the rate and extent of receptor cleavage. A low concentration of protease or in the presence of an inefficient protease, only a limited number of receptors would be activated. Although a limited number of activated receptors could transduce a signal, a measurable response requires activation of a minimum number of receptors at a certain rate.19 Additionally, the rate and extent of receptor activation required to elicit a specific response may be dependent on the response that is measured. For example, when receptor cleavage correlates with phosphoinositide hydrolysis, IP3 formation is proportional to the absolute amount of cleaved receptor, but the subsequent increase in cytosolic Ca2+ occurs only if IP3 is generated quickly enough to accumulate.44 However, this reasoning may not fully explain the phenomenon described given that FVIIa (10 nM), trypsin (1 nM), and PAR2 peptide agonist (1 μmol/L) treatments all result in a similar rate of IP3 hydrolysis, yet only trypsin and PAR2 peptide agonist, and not FVIIa, produced a clear increase in Ca2+ release (unpublished data of authors).

    Although the TF–VIIa complex is sufficient to induce cell signaling, it is unclear whether this binary complex functions as an efficient signaling unit in vivo. In many of the studies described here, high concentrations (10 to 100 nM) of FVIIa were required to obtain a measurable signaling response.15,21,26,42 It is likely that such high concentrations of FVIIa are needed to saturate TF rapidly, which may be essential for measuring the signaling response using a short-term assay, such as Ca2+ release or MAPK activation. In fact, low concentrations of FVIIa (5 to 10 nM) are shown to be capable of producing a pronounced response when signaling is analyzed using a long-term assay, such as gene expression28,38,39 However, in circulation most of the FVII is in zymogen form. FVIIa concentration in the plasma is 1% or less (100 pM) of the total circulating FVII (10 nM).45,46 Thus, it is crucial to show that traces of FVIIa in FVII are sufficient to induce cell signaling. However, it is pertinent to note here that because FVII bound to TF can be autocatalytically converted to FVIIa,47–49 most of the FVII bound to cell surface TF will be converted to FVIIa. Consistent with this notion, we found that plasma concentration of zymogen FVII added to fibroblasts39 and tumor cells38 induced Cyr61 and IL-8 gene expression, respectively, with a slight delay. The delay probably reflects the time required for autoactivation of FVII. When substrate FX is present at plasma concentrations, picomolar concentrations of FVIIa elicit a robust signal in cells expressing TF and PAR2.15 This suggests that the TF–VIIa-generated FXa is capable of signaling independent of thrombin. Riewald and Ruf found that the activation of FX by the TF–VIIa complex resulted in a much more robust signal than that induced by the TF–VIIa complex alone, free FXa, or FXa that was generated in situ by the intrinsic activation complex.42 These data, coupled with additional studies using a unique inhibitor (NAPc2, nematode anticoagulant protein C2) that preserves FXa activity in the complex while inhibiting free FXa and TF–VIIa proteolytic activity, revealed that the transient ternary TF–VIIa–Xa complex is a potent signaling unit in which FXa efficiently activates both PAR1 and PAR2.42 This finding supports the hypothesis that upstream coagulation protease signaling is mechanistically coupled to the initiation of the coagulation pathway. Although our data support the concept that the ternary complex of TF–VIIa–Xa is a more potent activator of PAR2 than the binary TF–VIIa complex, this difference is abolished when TF sites are saturated with FVIIa (Figure 2).38 It is currently difficult to conclude whether the ternary or the binary complex is the primary signaling unit in vivo. It is likely that both complexes play a role in vivo, and which complex is more active may depend on multiple factors, such as the levels of TF and PAR2/PAR1 expression, the availability of FX, and the localization/organization of these components on the cell membrane.

    Figure 2. A comparison of efficiency of TF–VIIa-mediated and TF–VIIa–Xa-mediated cell signaling. MDA-MB-231 cells were stimulated with varying concentrations of FVIIa in the presence or absence of substrate FX (175 nM). The signaling was evaluated in either IP3 hydrolysis assay (A) or IL-8 gene expression (B).

    Although it is apparent that TF–VIIa elicits cell signaling via activation of PAR2, it is unclear whether other cell membrane components participate in the signaling process. It is interesting to note that in some cases, even a high concentration of FVIIa fails to elicit a signaling response, even in cells expressing both functional TF and PAR2.24,50 It raises a possibility that other cell components may contribute or regulate TF–VIIa-induced cell signaling. In this context, it is important to note that Wiiger and Prydz31 recently demonstrated that the epidermal growth factor receptor is involved in the transduction of the TF–VIIa signal in HaCaT cells. However, the role of epidermal growth factor receptor in TF–VIIa-induced signaling still needs to be confirmed.

    TF Cytoplasmic Domain-Dependent Signaling

    In heterologous expression studies, the TF cytoplasmic domain is not clearly required for the TF–VIIa-induced cell signaling.24,26,34 However, it is unclear whether TF, as a true receptor, can transmit cell signaling via its cytoplasmic tail, or whether the cytoplasmic domain can regulate the protease-induced signaling. Analysis of human TF protein sequence revealed a consensus sequence for protein kinase C phosphorylation in the cytoplasmic domain, which is shown to be phosphorylated in response to phorbol esters.51 Mody and Carson52 showed, using a synthetic peptide corresponding to residues 245 to 263 of the human TF cytoplasmic domain and glioblastoma cell extract (as a source of kinase activity), that TF cytoplasmic domain is phosphorylated at multiple serine residues. Recent studies show that protein kinase C-dependent phosphorylation of Ser253 enhances subsequent Ser258 phosphorylation by a proline-directed kinase.53 A functional role for the TF cytoplasmic domain was documented in hematogenous metastasis. Deletion of the cytoplasmic domain54,55 or mutation of the cytoplasmic phosphorylation sites Ser253 and Ser25855,56 are shown to reduce the TF-induced metastasis. Abe et al57 showed that the TF cytoplasmic domain, independent of FVIIa, is responsible for the upregulation of VEGF in melanoma cells transfected with TF. However, others failed to confirm this finding.58 Recent studies with transgenic mice that lack the cytoplasmic domain of TF (but have normal coagulant function) indicate that the cytoplasmic domain of TF contributes to NF-B activation, pro-inflammatory cytokine production, and leukocyte recruitment after endotoxin challenge. However, further studies are needed to confirm that the TF cytoplasmic domain is directly responsible for the observed effects. Other studies suggest that the TF cytoplasmic domain contributes to cell signaling indirectly, ie, by modulating TF–VIIa-induced cell signaling. For example, deletion of the TF cytoplasmic domain is shown to impair TF–VIIa protease activity-induced reactive oxygen species production in monocytes. Elegant studies performed recently by Ahamed et al show that TF–VIIa–Xa activation of PAR2 induces TF cytoplasmic domain phosphorylation,59 and this phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of PAR2 signaling-mediated angiogenesis60 (Figure 2). At present, it is unclear whether this regulatory mechanism is specific for endothelial cells and the angiogenic process or if it also plays a role in modulating other PAR2 signaling-mediated events in other cell types.

    A Role for TF–VIIa Signaling in Pathophysiology

    It is widely accepted that TF, in addition to its role in coagulation, may contribute to various pathophysiological processes. However, it is unclear whether TF–VIIa-induced (or TF–VIIa–Xa-induced) cell signaling contributes directly to these biological processes, or whether cell signaling induced by coagulation proteases generated by TF–VIIa, such as thrombin in concert with an end product fibrin, fully accounts for the altered cellular processes. Recent studies suggest that both mechanisms are involved. The following discussion is limited to the potential role of direct TF–VIIa-induced cell signaling in a select few biological processes (Figure 3). For discussion on the role of TF in development, the reader is referred to a recent review.11

    Figure 3. TF–VIIa signaling mediated cellular effects and their contribution to various pathophysiological processes (see text for details).

    Inflammation

    It is well-established that inflammatory mediators activate coagulation by inducing TF expression on blood mononuclear cells and probably on vascular endothelium. In turn, expression of TF on these cells appears to regulate the inflammatory response. The observation that during sepsis the TF-initiated coagulation pathway induces a lethal inflammatory escalation even when fibrin formation and microthrombosis are effectively blocked via decreased thrombin generation61–65 suggests that TF–VIIa cell signaling induces a pro-inflammatory response. Additionally, inhibition of the TF–VIIa complex during septicemia significantly reduces the inflammatory response, as measured by decreased IL-6 and IL-8 plasma levels and reduced inflammatory changes in the lung, such as neutrophil infiltration and edema.63,66 In vitro studies67 also indicate that TF plays an important role in reverse transmigration of mononuclear phagocytes, a process that occurs during the resolution of acute inflammation. FVIIa binding to TF has been shown to augment the macrophage pro-inflammatory functions, such as the production of reactive oxygen species and the expression of major histocompatability complex class II and cell adhesion molecules, both in vivo and in vitro.68 Furthermore, recent studies using the mouse model of endotoxemia have shown that genetically modified mice expressing low levels of TF exhibited reduced IL-6 induction and increased survival compared with control mice.69 Although a deficiency of either PAR1 or PAR2 has no effect on inflammation or survival, a combination of thrombin inhibition and PAR2 deficiency reduces both inflammation and mortality similar to that observed in the low-TF mice.69 These data suggest that PAR1 and PAR2 may have partially redundant roles and contribute to the link between coagulation and inflammation. Together, these data imply that TF–VIIa-induced cell signaling via PAR2 may contribute to pro-inflammation. However, it is difficult to conclude from these results whether the pro-inflammatory signaling unit is TF–VIIa, TF–VIIa–Xa, or FXa, given that all of them are capable of activating PAR2. Furthermore, the reduced lipopolysaccharide-induced IL-6 expression and enhanced survival in the low-TF mice may be unrelated to the defect in TF–VIIa protease-induced signaling given that similar effects were observed in mice expressing TF that lacks the cytoplasmic domain and has normal coagulant function.70 However, one should exercise caution in interpreting the data from the latter studies because these studies were performed in mice with a mixed genetic background, which likely leads to a high degree of variability.

    Tumor Angiogenesis

    Hemostasis and angiogenesis are interrelated processes. Proteins generated by the hemostatic system are known to have regulatory effects on angiogenesis.71 TF-induced coagulation can indirectly support angiogenesis in several ways, such as the release of positive and negative regulators from activated platelets, thrombin signaling via endothelial cell PAR1, and the generation of provisional fibrin-rich matrix. Whether TF–VIIa signaling plays a direct role in the regulation of angiogenesis is not entirely clear. Overexpression of TF in tumor cells leads to the upregulation of the pro-angiogenic factor VEGF and the downregulation of the anti-angiogenic protein thrombospondin-1.72 However, Bromberg et al found no correlation between TF expression and VEGF.56 Recent studies have shown that TF–VIIa induces IL-8 production via PAR2 signaling, and the secreted IL-8 is capable of stimulating tumor cell migration and invasion in an autocrine fashion.38 IL-8 secreted by tumor cells was shown previously to regulate angiogenesis directly by enhancing endothelial cell survival, proliferation, and matrix metalloproteinase production.73 Surprisingly, although many cancer cell lines express functional PAR1, PAR2, and TF, these cells respond to either thrombin or FVIIa, but not to both in increasing IL-8 (unpublished data of authors). This observation suggests that other cell components, either on the cell surface or within the cell, provide further specificity for the protease-induced signaling.

    In cancer cells (unpublished data of the authors) and fibroblasts,39 TF–VIIa signaling upregulates the expression of CCN1, a novel matrix signaling protein that is a ligand to integrin v?3 on endothelium.74 Integrin v?3, an adhesion receptor known to be involved in signaling, regulates a number of cellular processes, including angiogenesis and tumor metastasis. The stimulatory effects of CCN1 on cell proliferation, migration, and survival via its interaction with various integrins are thought to be responsible for its role in angiogenesis and tumorigenesis.75 In addition to CCN1 and IL-8, TF–VIIa-induced cell signaling may also upregulate a number of other gene products, including uPAR,36 which plays a regulatory role in angiogenesis. Microarray analyses of MDA-MB-231 breast carcinoma cells exposed to FVIIa and a control vehicle show that FVIIa induces a set of genes whose products play a role in various steps of angiogenesis and tumor growth. The gene products include chemokines, cytokines, growth factors, cell adhesion proteins, and proteins involved in cell cycle control, apoptosis, and inflammation (unpublished data). Together, these data support the hypothesis that TF–VIIa cell signaling may play an important role in angiogenesis regulation (Figure 4).

    Figure 4. TF–VIIa signaling and angiogenesis. Binding of FVIIa to tumor cell TF activates a genetic program, which leads to upregulation of various chemokines, cytokines, and growth factors, including IL-8, CCN1, and VEGF. These secreted cytokines and growth factors from tumor cells promote endothelial cell proliferation and migration, leading to angiogenesis.

    Recent studies indicate a complex role for TF in angiogenesis. Belting et al60 show that genetic deletion of the TF cytoplasmic domain enhances PAR2-dependent angiogenesis, presumably in synergy with platelet-derived growth factor BB. In neonatal mice, the diameter of the superficial vascular plexus of TF–cytoplasmic domain-deleted mice was twice that observed in wild-type mice, indicating that the TF cytoplasmic tail negatively regulates in vivo angiogenesis during postnatal development. Furthermore, ocular tissues from diabetic patients display a colocalization of PAR2 and phosphorylated TF specifically on neovasculature. Overall, these observations suggest that phosphorylation of the TF cytoplasmic domain releases its negative regulatory control of angiogenesis. The role of the TF cytoplasmic domain as a negative regulator of physiological process has been further supported by the recent observation that TF expression suppresses 3?1-dependent migration on laminin 5, an effect that is reversed by PAR2-dependent phosphorylation of the TF cytoplasmic domain.76 Although these data support nonhemostatic roles for TF in angiogenesis and tumor metastasis, they do not fully explain the earlier studies that demonstrated that the TF–cytoplasmic domain contributes to cell migration,77 VEGF production,57 and tumor metastasis.54,55 The identified link between PAR2–TF and TF–integrins add yet another facet to the complex regulation of angiogenesis by TF–VIIa signaling.

    Tumor Metastasis

    The role of TF in metastasis is well-documented. TF expression has been correlated with malignant progression in several types of cancer.78–82 Experiments using mouse models have demonstrated that TF in tumor cells promotes hematogenous metastasis, and both the TF cytoplasmic domain and the TF–VIIa protease activity contribute to this metastasis.54–56,83 It is likely that the principle role for TF–VIIa protease activity in tumor metastasis is to generate thrombin and fibrin, which could play a direct role in tumor metastasis. Nonetheless, there is a possibility that TF–VIIa signaling may also contribute to tumor metastasis because it alters many cellular processes that are associated with tumor metastasis, such as angiogenesis. Furthermore, FVIIa induces the activation of both p42/44 MAPK and protein kinase B pathways, 2 pathways known to inhibit apoptosis.84,85 Resistance to apoptosis is a key factor in the survival of malignant cells. Defects in apoptosis or activation of anti-apoptotic pathways promote tumor growth and survival, which may be linked to tumor metastasis.86,87 Recent studies88,89 have shown that TF–VIIa activates anti-apoptotic signaling pathways in serum-deprived cells, thereby increasing their survival. Additionally, FVIIa was found to inhibit apoptosis induced by the loss of adhesion.88 Therefore, TF–VIIa-induced cell survival, in addition to other TF–VIIa-induced cellular processes, may contribute to tumor growth and metastasis. However, it should be noted that the evidence for the anti-apoptotic effect of FVIIa is limited to cells that were transfected to overexpress TF. Confirmation of the anti-apoptotic effect of FVIIa in tumor cells and/or stromal cells surrounding a tumor is essential before strong mechanistic conclusions of how TF–VIIa contributes to tumor growth and metastasis can be drawn.

    Although it is logical to assume that tumor TF contributes to tumor growth and metastasis through TF–VIIa activation of PAR2 and/or PAR1, currently there is no evidence to support this. Both PAR1 and PAR2 are coexpressed in tumor cells and cells surrounding a tumor in the tumor microenvironment.90 Although many studies document the importance of thrombin activation of PAR1 in tumor cells in metastasis,91–97 little is known regarding the role of tumor cell PAR2 in metastasis, let alone the importance of TF–VIIa-induced activation of PAR2. Recently, Shi et al98 showed that both PAR1 and PAR2 are involved in tumor metastasis and PAR2 effects on tumor cell migration and metastasis are thrombin-dependent. At present, it is unclear whether PAR2 in metastasis is activated indirectly by thrombin or directly by a protease with trypsin-like activity, such as FVIIa. Studies addressing the role of PAR1 and PAR2 of host tissues in tumor metastasis show both PAR1 and PAR2 deficiency have no effect on tumor metastasis.99 In contrast, genetic deficiency in platelet production or activation protected mice against metastasis in hematogenous metastasis model system.99 These data raise a valid question whether PAR1 and PAR2 activation in endothelial or inflammatory cells contribute to tumor metastasis.

    Wound Healing

    The epidermis is a rich source for TF.1 Most wounds to the skin will invariably cause leakage of blood from the damaged blood vessels. This leakage allows the formation of a fibrin clot, which temporarily shields the wound and protects the denuded wound tissues, and it also releases various growth factors and cytokines from degranulating platelets that trigger the repair process immediately after injury.100,101 The blood leakage at the wound site will also allow FVIIa binding to TF on keratinocytes, raising a possibility for a role for TF–VIIa-induced cell signaling in the wound repair. Consistent with this scenario, Camerer et al28 showed that the interaction of FVIIa with TF on human keratinocytes upregulated a number of genes that are shown to be involved in the early steps of wound healing. They include transcription regulators (c-fos, EGR-1, ETR101, BTEB2, c-myc, fra-1, and tristeraproline), growth factors (amphiregulin, hbEGF, CCN2, and FGF-5), pro-inflammatory cytokines (IL-1?, IL-8, LIF, and MIP2), proteins involved in cellular reorganization/migration (RhoE, uPAR, and collagenases 1 and 3), and others (plasminogen activator inhibitor-2, cyclophilin, GADD45, Jagged 1, and prostaglandin E2 receptor). Because FVIIa binding to TF would occur immediately on wounding, TF–VIIa signaling may provide an early signal in the wound repair process. The expression pattern of TF–VIIa-induced genes suggests that TF–VIIa signaling may contribute to various steps in wound healing. TF–VIIa signaling may also play a role in wound healing indirectly through elaboration of gene products that in turn could induce cell signaling appropriate for the wound repair process. In this context, TF–VIIa-induced expression of CCN1 and CCN2 in keratinocytes and fibroblasts are noteworthy.28,39 CCN1 is inducibly expressed in granulation tissue during wound repair and activates genes that play multiple and coordinated roles in the wound healing process, including angiogenesis, inflammation, and extracellular matrix remodeling.102 Similarly, CCN2 contributes to wound healing via selective modulation of fibroblasts proliferation and changes to gene expression.103

    Atherosclerosis and Smooth Muscle Cells

    It is well-established that exposure of TF to circulating blood on rupture of atherosclerotic plaque plays an important role in the pathogenesis of thrombus formation at sites of plaque rupture, resulting in acute coronary events and myocardial infarction.104–107 Recent studies indicate that TF–VIIa signaling may play a role in the pathogenesis of atherosclerosis. TF–VIIa has been shown to be a strong chemotactic stimulus for smooth muscle cells,108 and overexpression of tissue factor pathway inhibitor in smooth muscle cells was shown to attenuate the TF–VIIa-induced cell migration.109 Consistent with this in vitro observation, overexpression of tissue factor pathway inhibitor was found to attenuate vascular remodeling in a murine model system.110 TF–VIIa-induced cell signaling has also been shown to promote platelet-derived growth factor BB-induced cell migration in fibroblasts.111 Additionally, TF–VIIa-induced cell signaling recently has been shown to lead to smooth muscle cell proliferation.112 Although our recent data113 using fibroblasts support this observation, the proliferative effect of TF–VIIa-induced cell signaling was very modest. Moreover, other studies failed to demonstrate the proliferative effect of TF–VIIa signaling.29,89,114 Therefore, further studies are needed to determine whether TF–VIIa-induced cell proliferation contributes to the pathogenesis of atherosclerosis. Because TF–VIIa-induced cell signaling in various cell types was shown to induce a number of gene products that are relevant to cell proliferation and migration, it is possible that TF–VIIa signaling may also produce similar effects in TF-expressing cells in the atherosclerosis plaques. It is of particular interest to note that TF–VIIa signaling upregulates the expression of CCN1 and CCN2 in fibroblasts.39 Similar to TF expression, CCN1 mRNA is undetectable in normal blood vessels but overexpressed in atherosclerotic lesions, primarily in vascular smooth muscle cells.115 In atherosclerosis, high levels of CCN2 expression is thought to be responsible for extracellular matrix accumulation and thus progression of atherosclerotic lesions.116 Similar to CCN2, CCN1 expression was shown to be upregulated in atherosclerotic lesions of apoE–/– mice117 and human atherosclerotic lesions.118 Because recent studies show extrahepatic synthesis of FVII in human atherosclerotic vessels,119 it is possible that TF–VIIa may induce the expression of CCN1 and CCN2 within the plaque, which could accelerate intimal thickening by promoting proliferation and migration of fibroblasts and smooth muscle cells. Further, removal or retraction of endothelial cells of atherosclerotic plaques would expose CCN1 and CCN2 in the underlying subendothelial matrix to which activated platelets and monocytes could adhere.117,120 This, in combination with TF–VIIa-induced fibrin clot, could lead to an acute arterial occlusion on rupture of the atherosclerotic plaque. Paradoxically, increased expression of CCN2 could also play a beneficial role. For example, increased expression of CCN2 along the fibrous cap may reduce the risk of plaque rupture by stabilizing the fibrous cap with extracellular matrix.

    Summary and Future Directions

    Recent studies suggest that TF plays a nonhemostatic role in many biological processes, and at least some of these effects are mediated via TF–VIIa-induced cell signaling. It is now established that TF–VIIa induces cell signaling via activation of PAR2 and that the ternary complex of TF–VIIa–Xa is a more efficient signaling mediator than the binary TF–VIIa complex, particularly at low concentrations of FVIIa. However, it is currently unclear whether the ternary and the binary complexes activate the same signaling pathways or if each has its own specificities. Because TF–VIIa complex primarily activates PAR2 while TF–VIIa–Xa activates both PAR1 and PAR2, it is expected that there would be some distinction between the signaling mediated by these complexes. Identification of such differences would further reinforce the importance of TF–VIIa signaling. In addition to PARs, other cell surface components, such as integrins, proteoglycans, and growth factor receptors, may also be involved in transmitting or regulating TF–VIIa signaling. Analysis of other potential cell surface receptors that may mediate TF–VIIa signaling is an important area requiring further study. The ability of FVIIa to induce cell signaling is dependent not only on the availability of PARs and TF but also on their spatial proximity. A spatial disconnection between TF and PAR2 may explain why FVIIa fails to induce signaling in some cells, even if they express both TF and PAR2. Currently, there is no information on how the spatial organization of participating signaling components regulates TF–VIIa signaling. This is also another important area in need of further investigation. Additionally, analysis of if and how phosphorylation of the TF cytoplasmic domain regulates TF–VIIa protease-induced signaling also requires future attention. Until now, the TF–VIIa-induced cell signaling studies are primarily limited to in vitro cell model systems and there is no convincing evidence that TF–VIIa signaling actually plays a role in vivo. Thus, the main challenge for future investigators is to establish the importance of TF–VIIa-induced cell signaling in pathophysiological processes in which multiple protease-induced signaling pathways operate simultaneously and redundantly in PAR-mediated signaling. The development of specific inhibitors that suppress TF–VIIa signaling but not TF–VIIa coagulant function or vice versa will provide unique opportunities to design specific drugs that may have therapeutic value in the treatment of diseases that are often associated with aberrant expression of TF.

    Acknowledgments

    The authors are thankful to Drs Lars Petersen, Brit Sorensen, and Mirella Ezban at Novo-Nordisk, Denmark, for their collaboration on the work related to TF–VIIa signaling. The authors’ work related to this article was supported by National Institutes of Health grants HL65550 and HL58869, and grants from American Heart Association (National and Texas affiliate).

    Received September 28, 2004; accepted November 12, 2004.

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